Regulation of the cell cycle is important in homeostasis of both cells and organisms (e.g., mammalian cells or mammals). Disruptions in the normal regulation of the cell cycle can occur, for example, in tumors which proliferate uncontrollably, in response to DNA damage (e.g., ionizing radiation) to the cell or organism, and under conditions of stress (e.g., oxidative stress) in the cell or organism.
The p53 tumor suppressor protein exerts anti-proliferative effects, including growth arrest, apoptosis, and cell senescence, in response to various types of stress, e.g., DNA damage (Levine, 1997; Giaccia and Kastan, 1998; Prives and Hall, 1999; Oren, 1999; Vogelstein et al., 2000). Inactivation of p53 function appears to be critical to tumorigenesis (Hollstein et al., 1999). Mutations in the p53 gene have been shown in more than half of all human tumors (Hollstein et al., 1994). Accumulating evidence further indicates that, in the cells that retain wild-type p53, other defects in the p53 pathway also play an important role in tumorigenesis (Prives and Hall, 1999; Lohrum and Vousden, 1999; Vousden, 2000). The molecular function of p53 that is required for tumor suppression involves its ability to act as a transcriptional factor in regulating endogenous gene expression. A number of genes which are critically involved in either cell growth arrest or apoptosis have been identified as p53 direct targets, including p21CIP1/WAF1, Mdm2, GADD45, Cyclin G, 14-3-3F, Noxa, p53AIP1, PUMA and others (Nakano and Vousden, 2001; Yu et al., 2001; Oda et al., 2000a, 2000b; El-Deriry et al., 1993; Wu et al., 1993; Barak et al., 1993; Kastan et al., 1992; Okamoto and Beach, 1994).
p53 is a short-lived protein whose activity is maintained at low levels in normal cells. Tight regulation of p53 is essential for its effect on tumorigenesis as well as maintaining normal cell growth. The precise mechanism by which p53 is activated by cellular stress is not completely understood. It is generally thought to involve primarily post-translational modifications of p53, including phosphorylation and acetylation (reviewed in Appella and Anderson, 2000; Giaccia and Kastan, 1998). Early studies demonstrated that CBP/p300, a histone acetyl-transferase (HAT), acts as a coactivator of p53 and potentiates its transcriptional activity as well as biological function in vivo (Gu et al., 1997; Lill et al., 1997; Avantaggiati et al., 1997). Genetic studies have also revealed that p300 mutations are present in several types of tumors, and that mutations of CBP in human Rubinstein-Taybi syndrome as well as CBP knockout mice lead to higher risk of tumorigenesis, further supporting an important role for this interaction in the tumor suppressor pathway (reviewed in Goodman and Smolik, 2000; Gile et al., 1998; Kung et al., 2000; Gayther et al., 2000). Significantly, the observation of functional synergism between p53 and CBP/p300 together with its intrinsic HAT activity led to the discovery of a novel FAT (Transcriptional factor acetyl-transferase) activity of CBP/p300 on p53 which suggests that acetylation represents a general functional modification for non-histone proteins in vivo (Gu and Roeder, 1997) which has been shown for other transcriptional factors (reviewed in Kouzarides, 2000; Sterner and Berger, 2000; Muth et al., 2001).
p53 is specifically acetylated at multiple lysine residues (Lys 370, 371, 372, 381, 382) of the C-terminal regulatory domain by CBP/p300. The acetylation of p53 can dramatically stimulate its sequence-specific DNA binding activity, perhaps as a result of an acetylation-induced conformational change (Gu and Roeder, 1997; Sakaguchi et al., 1998; Liu et al., 1999). By developing site-specific acetylated p53 antibodies, CBP/p300 mediated acetylation of p53 was confirmed in vivo by a number of studies (reviewed in Chao et al., 2000; Ito et al., 2001). In addition, p53 can be acetylated at Lys320 by another HAT cofactor, PCAF, although the in vivo functional consequence needs to be further elucidated (Sakaguchi et al., 1998; Liu et al., 1999; Liu et al., 2000). Steady-state levels of acetylated p53 are stimulated in response to various types of stress (reviewed in Ito et al., 2001).
Recently, by introducing a transcription defective p53 mutant (p53Q25S26) into mice, it was found that the mutant mouse thymocytes and ES cells failed to undergo DNA damage-induced apoptosis (Chao et al., 2000; Jimenez et al., 2000). Interestingly, this mutant protein was phosphorylated normally at the N-terminus in response to DNA damage but could not be acetylated at the C-terminus (Chao et al., 2000), supporting a critical role of p53 acetylation in transactivation as well as p53-dependent apoptotic response (Chao et al., 2000; Luo et al., 2000). Furthermore, it has been found that oncogenic Ras and PML upregulate acetylated p53 in normal primary fibroblasts, and induce premature senescence in a p53-dependent manner (Pearson et al., 2000; Ferbeyre et al., 2000). Additionally acetylation, not phosphorylation of the p53 C-terminus, may be required to induce metaphase chromosome fragility in the cell (Yu et al., 2000). Thus, CBP/p300-dependent acetylation of p53 may be a critical event in p53-mediated transcriptional activation, apoptosis, senescence, and chromosome fragility.
In contrast, much less is known about the role of deacetylation in modulating p53 function. Under normal conditions, the proportion of acetylated p53 in cells remains low. This may reflect the action of strong deacetylase activities in vivo. The acetylation level of p53 is enhanced when the cells are treated with histone deacetylase (HDAC) inhibitors such as Trichostatin A (TSA). These observations led to identification of a HDAC1 complex which is directly involved in p53 deacetylation and functional regulation (Luo et al., 2000; Juan et al., 2000). PID/MTA2, a component of the HDAC 1 complex, acts as an adaptor protein to enhance HDAC 1-mediated deacetylation of p53 which is repressed by TSA (Luo et al., 2000). In addition, Mdm2, a negative regulator of p53, actively suppresses CBP/p300-mediated p53 acetylation, and this inhibitory effect can be abrogated by tumor suppressor p19ARF. Acetylation may have a critical role in the p53-MDM2-p19ARF feed back loop (Ito et al., 2001; Kobet et al., 2000).
The Silent Information Regulator (SIR) family of genes represents a highly conserved group of genes present in the genomes of organisms ranging from archaebacteria to a variety of eukaryotes (Frye, 2000). The encoded SIR proteins are involved in diverse processes from regulation of gene silencing to DNA repair. The proteins encoded by members of the SIR2 gene family show high sequence conservation in a 250 amino acid core domain. A well-characterized gene in this family is S. cerevisiae SIR2, which is involved in silencing HM loci that contain information specifying yeast mating type, telomere position effects and cell aging (Guarente, 1999; Kaeberlein et al., 1999; Shore, 2000). The yeast Sir2 protein belongs to a family of histone deacetylases (reviewed in Guarente, 2000; Shore, 2000). The Sir2 homolog, CobB, in Salmonella typhimurium, functions as an NAD (nicotinamide adenine dinucleotide)-dependent ADP-ribosyl transferase (Tsang and Escalante-Semerena, 1998).
The Sir2 protein is a deacetylase which uses NAD as a cofactor (Imai et al., 2000; Moazed, 2001; Smith et al., 2000; Tanner et al., 2000; Tanny and Moazed, 2001). Unlike other deacetylases, many of which are involved in gene silencing, Sir2 is insensitive to histone deacetylase inhibitors like trichostatin A (TSA) (Imai et al., 2000; Landry et al., 2000a; Smith et al., 2000).
Deacetylation of acetyl-lysine by Sir2 is tightly coupled to NAD hydrolysis, producing nicotinamide and a novel acetyl-ADP ribose compound (1-O-acetyl-ADP-ribose) (Tanner et al., 2000; Landry et al., 2000b; Tanny and Moazed, 2001). The NAD-dependent deacetylase activity of Sir2 is essential for its functions which can connect its biological role with cellular metabolism in yeast (Guarente, 2000; Imai et al., 2000; Lin et al., 2000; Smith et al., 2000). Mammalian Sir2 homologs have NAD-dependent histone deacetylase activity (Imai et al., 2000; Smith et al., 2000). Most information about Sir2 mediated functions comes from the studies in yeast (Gartenberg, 2000; Gottschling, 2000).
Among Sir2 and its homolog proteins (HSTs) in yeast, Sir2 is the only protein localized in nuclei, which is critical for both gene silencing and extension of yeast life-span (reviewed in Guarente, 2000). Based on protein sequence homology analysis, mouse Sir2α and its human ortholog SIRT1 (or human Sir2α or hSir2) are the closest homologs to yeast Sir2 (Imai et al., 2000; Frye, 1999, 2000) and both exhibit nuclear localization (FIG. 7C). Homologues of Sir2 have been identified in almost all organisms examined including bacteria, which has no histone proteins (reviewed in Gray and Ekstrom, 2001; Frye, 1999; 2000; Brachmann et al., 1995). For this reason it is likely that Sir2 also targets non-histone proteins for functional regulation (Muth et al., 2001).
The S. cerevisiae Sir2 is involved in DNA damage responses (Martin et al., 1999; McAinsh et al., 1999; Mills et al., 1999). In mammalian cells, one of the primary mediators of the DNA damage response is the p53 protein (Levine, 1997; Oren, 1999; Vogelstein et al., 2000). Following DNA damage, the p53 protein is protected from rapid degradation and acquires transcription-activating functions, these changes being achieved largely through post-translational modifications (Abraham et al., 2000; Canman et al., 1998; Chehab et al., 1999; Sakaguchi et al., 1998; Shieh et al., 2000; Siliciano et al., 1997). Transcriptional activation of p53 protein in turn upregulates promoters of a number of genes including p21WAF1 (el-Deiry et al., 1993) that promotes cell cycle exit or death-inducing proteins like PIDD (Lin et al., 2000).
The p53 protein is phosphorylated in response to DNA damage (Siliciano et al., 1997). There are at least 13 different residues both at the N and C terminal portions of p53 protein that are phosphorylated by various kinases (Appella and Anderson, 2000). For example, the ATM and ATR proteins phosphorylate p53 at residue Ser15 (Khanna et al., 1998; Siliciano et al., 1997; Tibbetts et al., 1999) and Chk1/2 kinases at residue Ser20 (Chehab et al., 1999; Shieh et al., 2000).
Modification of Ser15 is important for the functional activation of the p53 protein. Phosphorylation of Ser15 may increase the affinity of the p300 acetylase for p53 (Dumaz and Meek, 1999; Lambert et al., 1998).
p53 is acetylated in vitro by p300 at Lys 370-372, 381 and 382 (Gu and Roeder, 1997). In response to DNA damage, p53 is also acetylated in vivo at Lys 373 and Lys 382 (Abraham et al., 2000; Sakaguchi et al., 1998). Other factors that can affect acetylation of p53 include MDM2 protein, which is involved in the negative regulation of p53 (Oren, 1999) and can suppress acetylation of p53 protein by p300 (Ito et al., 2001; Kobet et al., 2000). While acetylation by p300 and deacetylation by the TSA-sensitive HDAC1 complex (Luo et al., 2000) have been shown to be important in regulation of p53 protein activity, the remaining factors responsible for its regulation as a transcription factor remain elusive.
Analogs of NAD that inhibit endogenous ADP-ribosylases reduce induction of p21WAF1 in response to DNA damage and overcome p53-dependent senescence (Vaziri et al., 1997). In addition, p53 protein can bind to the NAD-dependent poly-ADP-ribose polymerase.
The SIR complex in Saccharomyces cerevisiae was originally identified through its involvement in the maintenance of chromatin silencing at telomeres and at mating type loci. It is composed of four components, Sir1p, Sir2p, Sir3p, and Sir4p, that normally reside at yeast telomeres. In response to DNA damage, the SIR complexes relocate to the site of double-stranded breaks where they participate in the repair of the lesions by non-homologous end joining This DNA damage response is dependent on the function of the MEC1/RAD9 DNA checkpoint pathway. MEC 1 is a homolog of the ATM protein that coordinates the DNA damage response in mammalian cells, in part by triggering the cascade of events that lead to the stabilization of the p53 protein (Canman et al., 1998). Another major function of Sir2, gene silencing, is closely tied to the regulation of lifespan in S. cerevisiae (Guarente, 1999).
Double-strand breaks in the genome of mammals invoke a cascade of signaling events that ultimately cause phosphorylation and subsequent stabilization of p53 protein. In addition, these strand breaks lead to activation of p53 protein as a transcription factor. This activation may be due largely to its acetylation (Gu and Roeder, 1997; Sakaguchi et al., 1998). The resulting stabilized, activated p53 protein contributes to the upregulation of cyclin-dependent kinase inhibitors such as p21 WAF1 and hence to the cytostatic effects of p53. Alternatively, depending on the cellular background or degree of damage, the apoptotic effects of p53 may predominate through its ability to induce expression of pro-apoptotic proteins such as PIDD (Lin et al., 2000). These various phenomena indicate that specific components of the machinery that monitors the integrity of the genome are clearly able to alert p53 to the presence of genetic damage, leading to its functional activation. Conversely, in the event that damage has been successfully repaired, signals must be conveyed to p53 in order to deactivate it. Thus, a cell cycle advance that has been halted by p53 to enable repair to proceed should be relieved following completion of repair, enabling the cell to return to its active growth state. For this reason, the inactivation of p53 becomes as important physiologically as its activation.
In light of this information, modulators of Sir2 and/or p53 activity would be useful in modulating various cellular processes including, e.g., repair of DNA damage, apoptosis, oncogenesis, gene silencing and senescence, inter alia.